With an improvement in living standards, people care more and more about their health, safety and living conditions, which relies on analysis and tests, including medical clinical testing, food safety testing, environmental quality testing, etc. Those testing methods of chemical analysis and biochemical analysis are developed from the previous manual experimental methods to automatic analysis tests by means of automatic analytical instruments and on-site tests (e.g. POCT, point of care testing), greatly reducing the workload of the analysis operators. According to the analysis reagent types, the analysis tests can be divided into two methods: tests by means of liquid reagent and dry chemical reagent. By using the method of the liquid reagent (the wet chemistry), a sufficient accuracy and precision can be obtained, but it requires expensive instruments and professional personnel. In addition, it needs complicated operations and cannot obtain results quickly, which is not suitable for on-site tests (e.g. the demands for testing in hospital emergency rooms, and a small quantity of samples for tests). By contrast, the method of the dry chemical reagent has advantages in easy operation, rapid measurement, flexibility and no need for professional personnel which remedies the deficiencies of the wet chemistry well. The accuracy and precision of the dry chemical reagent are close to the testing method of the wet chemistry. Particularly, the dry chemical test-strip (paper), used in the dry chemistry, is widely used in the fields of medical clinical testing, food safety testing, and environmental quality testing, offering a great convenience. Relative to the traditional “wet chemistry” (i.e., solution chemistry), the “dry chemistry” is a method that the substance to be tested directly reacts with the dry reagent powder solidified on the carrier, with the liquid in the tested samples serving as a reaction medium. The greatest difference between the dry chemistry and the traditional wet chemistry lies with the different media involved in the chemical reactions. With the developments of separation, purification and storage of enzymes of biochemistry, and an advancement in the technology of sensors, photometers and electrodes, as well as the popularity of computer applications, the dry chemical technology has made a great progress in the past 20 years. The dry chemical test strip (paper) is now widely used in fields of medical clinical testing, food safety testing, and environmental quality testing, for which the methods of electrochemistry and colorimetry are commonly used.
The electrochemistry, as described in CN101762628A, entitled “Dry enzyme method biochip/test strip”, is made with the dry enzyme work electrode, the reference electrode, the testing liquid diffusing reticular membrane, the hydrophobic insulation paint on the PET substrate, and the testing result signals need to be read by electrochemical instruments.
The dry chemical test strip of colorimetry developed earlier, from the initial one-layer structure to two-layer structure, three-layer structure and the latest multilayer structure.
The initial one-layer structure, such as the pH strip for testing the acidity and alkalinity and the potassium-iodide-starch test strip for measuring oxides, both are mainly used in qualitative experiments, with semi-quantitative values at most.
The simplest reagent carrier for the biochemistry analysis, developed on the basis of the one-layer structure, is the two-layer structure. A cellulose sheet of the reagent layer is provided on the supporting layer, i.e., plastic substrate, with all the reagents having a pre-solid phase in the cellulose sheet. The urine biochemical analysis reagent strip is a common two-layer structure, as described in CN1888900A, entitled “PH Sectional sequencing 13 item urine test paper”. The thirteen test items are assigned to thirteen different reagent strips affixed on the plastic substrate. The composition of urine to be measured reacts directly with the reagents having a pre-solid phase in fee cellulose sheet to calculate the concentration of the composition to be measured by measuring color intensity changes through the reflection photometer. This structure can only be used for qualitative or semi-quantitative measurements, and the result of this strip also needs to be read by an instrument, with a narrow linear range of the measurement. Experimental results will be impacted in a circumstance of drug involvement, which limits its applications in other quantitative fields.
A porous membrane filtration layer is added on the reagent layer to form a three-layer structure membrane. The function of porous membrane filtration layer is to filter out impurities in the samples, protecting the reagent layer. The reagent strip for measuring glucose by micro method is a common three-layer structure. The optical measuring path of the reagent carrier in the three-layer structure is implemented by using the transparent plastic substrate, rather than passing through the top filter layer. As a result, the influence of the interfering composition in the samples is eliminated to ensure the stability and accuracy of the composition to be measured. For example, colorimetric blood glucose test strip preparing and supporting instruments are disclosed in CN1225450A, entitled “Test paper and analyte measuring tip”.
There is only one reagent layer in the three-layer structure. However, there are many analytical tests in which all the reagents cannot be mixed in advance, while some complex testing solution needs to be preheated, which limits the application fields of the dry chemical test strip (paper) with the three-layer structure. In order to overcome the defects of the three-layer structure, the current latest technology—a multilayer film method, i.e., multiple layers of membranes reagent carrier of the dry chemistry is provided. The multilayer film method can mainly be divided into longitudinal method and transverse method, according to the fabrication process. The first method is a multilayer film dry method, which is represented by Johnson & Johnson and Fujifilm corporations, applying the reagent layer, the auxiliary reagent layer, the light diffusing layer, the distributing layer and other reagents on the transparent support layer using coating technology of photographic film, so as to measure the optical density changes from the opposite side of the support layer, as disclosed in U.S. Pat. No. 5,508,173. The second method relates to the dry chemistry products of Roche as disclosed in U.S. Pat. No. 4,604,264, applying the reagent on the multi-silk fiber or fabric, contacting and reacting with the upper reagent layer after whole blood passing through the glass fiber for being filtered transversely; and then measuring the optical density changes by the transparent film which covers at the top.
Those dry chemical test strips (paper) described above are difficult to be quantitative. Further, some of the test strips have a complicated production process, and high requirements for materials and equipment. Some of them may cause some defects, such as a few bubbles and excess liquid, etc. easily generated at the sample adding end, causing possible interferences to the samples when the samples are added and measured on the same side, and temperature and time controls required for some testing items. Furthermore, the current dry biochemical analyzer is a large-scale equipment, with all the biochemical testing items integrated in a single machine, such as VITROS series of Johnson & Johnson (VITR0S-25, VITR0S-950), a semi-automatic and fully automatic dry biochemical analyzer and biochemical reagent, wherein VITR0S-250 and VITR0S-950 are mainly used in hospitals, and it is inconvenient to start the machine in case of only few samples. In case of emergencies on site such as, acute abdomen, an immediate test is urgently needed, and the quantitative test strip can meet the instant needs, which is of great significance for on-site testing.
In order to realize the rapid and accurate quantitative measurements and analysis on site, the sample liquid, is rapidly tested quantitatively on site, by artificial controls of the concentration distribution of reagent on the dry chemical test strip (paper). The liquid to be tested is realized to be diffused and permeated in the strip (paper), similar to the principle of chemical titration.
The method of CN2564584, entitled “Detecting plate of bacterial medicine sensitivity experiment for detecting minimum bacterium-inhibiting concentration of antibiotics” is using many different quantitative lands of antibiotic concentration gradient filter membrane strips fixed on the substrate. The feature is that the concentration of antibiotic on the plastic carrier exponentially and discontinuously decreases in the longitudinal direction, with the same concentration gradient method as the transverse direction (as shown in FIG. 5).
The method of CN201309942, entitled “Comb-shaped medicament susceptibility test scrip” is somewhat similar to the method of CN2564584. The feature is that the concentration of antibiotic on the comb teeth of the plastic carrier exponentially and discontinuously decreases in the longitudinal direction, with the concentration in the transverse direction (as shown in FIG. 6).
The method of CN1667414, entitled “Stepped concentration gradient detecting bar and preparing process thereof” is that the earner is divided into a plurality of equal areas on the same side of the carrier and the drag blocks having the set shape and area are applied symmetrically at the centers of each equal area. The dosage of each drug block changes with gradients of one or more segments, i.e. the drag blocks in different segments have different concentrations. But each drag block in the same segment has the same coating thickness and concentration. The size of each drug block increases or decreases sequentially with gradient, characterized in that the distribution of the concentration gradient is determined by the areas of the antibiotic agent coated on the substrate i.e., the concentration of the antibiotic agent is presented with a discontinuous gradient distribution longitudinally in the strip (paper) (as shown in FIG. 8), while the concentration of antibiotic agent is presented with a peak distribution transversely in the strip (paper) (as shown in FIG. 7). This method is also used in U.S. patent applications US20130244316A1, entitled “Paper strip for determining minimum inhibitory concentrations of antibiotics” and U.S. Pat. No. 4,778,758“Device for susceptibility testing of microorganisms”.
The patents as above realize the gradient distribution of antibiotic agent, mainly applied in the microbial drug sensitivity test, which is a test of microbial resistance. The distribution of the concentration of the antibiotic agent changing with gradient is formed on the medium surface by diffusing the antibiotic in the medium during the process of the experiment. The bacterial minimum inhibitory concentration (MIC value, Minimal inhibitory concentration) can then be read after cultivation, and thus the result cannot be rapidly obtained on site.